Genomic integrity depends upon the efficient repair of DNA base damage that would otherwise cause mutagenic and cytotoxic effects. The proposed integrated and synergistic structural (Scripps) and biochemical (SUNY) characterizations aim to define the structural biochemistry for DNA base repair (BR) by comparative studies of protein: DNA and protein: protein complexes acting in initiation of the major base damage response systems. We will evaluate DNA Base Excision Repair (BER) enzymes, which initiate repair pathways by recognizing and removing damaged bases and abasic sites, in concert with studies on direct base damage reversal by single enzymes and on other mechanisms for removal of non-classical bases from the nucleotide pool. We will address three fundamental hypotheses: 1) the self-assembly of DNA base repair complexes involves experimentally definable conformational controls, 2) initial committed steps in multi-step repair pathways generate stable intermediates or product complexes that create platforms influencing subsequent steps, and 3) direct damage reversal enzymes are coordinated by binding other components rather than by forming stable complexes with DNA products. Our pyramid approach involves beginning with multiple target enzymes in each aim and then progressively focusing on the most informative proteins. This strategy of pursuing multiple enzymes for each DNA BR class is thus aimed at efficiently achieving a unified understanding. We wiII be guided in the optimization of target complexes for each of the five Specific Aims by the rapidly emerging genetic and biochemical results on BR, including those from our strategically chosen collaborators. This research will test unifying hypotheses relevant to base repair enzyme activities and functions in genomic integrity, including the overall proposition that repair pathway progression and coordination are an emergent property of the structural chemistry for individual protein: damaged DNA base complexes. Comparative experiments among BR enzymes and species wiII test hypotheses from individual structures and better characterize structural motifs and chemical mechanisms in the most timely and cost effective fashion. This research aims to provide fundamental knowledge on DNA BR related complexes that will define their roles in the regulation of genome fidelity and the mechanisms w hereby loss of the functions of these coordinated complexes may lead to inheritable genetic defects and the initiation of cancer.